Immunization against dental caries with glucosyltransferase antigens

ABSTRACT

Immunization of animals with preparations containing more purified forms of glucosyltransferase (GTF) results in the presence of antibody in saliva demonstrable by functional inhibitions of enzyme activity and binding of radioactive enzyme. Serum antibody was also present. Immunized groups of animals had lower mean caries scores than comparably sham-immunized or nonimmunized control groups. Local immunization with GTF of serotype c or g or a Streptococcus mutans reduces the colonization, caries, and lesions caused by infection with S. mutans of serotype g (strain 6715) or c, or with serotype g or c, or with serotype a or g, respectively.

BASIS OF WORK

The invention described and claimed in this application has beendeveloped, in whole or in part, under NIDR Grant No. DE-000 24, DE-04733and DE-70122 and Contract No. DE-42438 of the National Institute ofHealth, Department of Health, Education and Welfare.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

This invention relates to the prevention of dental caries, and moreparticularly to the immunization against caries employing the glucosyltransferase (dextransucrase) enzyme (GTF) involved in the synthesis ofthe water-insoluble extracellular polysaccharide from sucrose by theaction of cariogenic bacteria. More specifically, it relates to methodsof obtaining more highly purified glucosyl transferase enzyme. Itfurther relates to cross protection via immunization with serotype cGTF, or with serotype g CTF or with serotype a GTF.

(2) Description of the Prior Art

Dental caries is the most prevalent infectious disease in the westernworld, and the cost of its treatment exceeds that of any singlebacterial infection.

Studies have indicated that the formation of carious lesions on teeth isrelated to the interaction between carbohydrates (notably sucrose) inthe diet and specific bacteria on tooth surfaces. The cariogenicbacteria, predominantly streptococci, adhere to the surfaces of teeth bysynthesizing extracellular polysaccharides from sucrose. Streptococcusmutans is the principle organism associated with the dental caries inhumans and will also produce disease in rodents and in primate models.O'Brien, T. C. 1976. Introduction and rationale for continuedinvestigations on a vaccine as an approach to dental caries prevention.Immunology Abstracts (Special Issue):3. Gibbons, R. J. and J. van Houte.1975. Bacterial adherence in oral microbial ecology. Ann. Rev.Microbiol. 29:19. These organisms ferment carbohydrate moieties whichresults in the production of acid leading to demineralization of thetooth enamel.

These polysaccharides, which are generally either polyglucans orpolyfructans (levan), "glue" the bacterial cells together and help themadhere to the teeth. The polysaccharides thus promote action between thebacteria and further sucrose ingested by the host animal and therebyfacilitate the formation of further polysaccharides. Moreover, theextracellular polysaccharides thus produced are believed to playsignificant roles in plaque formation and in the consequent developmentof caries. The polysaccaride polymers are synthesized from sucrose by agroup of extracellular and cell-associated consitutive enzymescollectively called glucosyltransferase (GTF).

At least two types of GTF enzymes have been described on the basis ofthe product synthesized. One type of GTF enzyme(s) will synthesizepredominantly water-insoluble glucose polymers (called mutan).Guggenheim, B. 1970. Enzymatic hydrolysis and structure ofwater-insoluble glucan produced by glucosyltransferases from a strain ofStreptococcus mutans. Helv. Odontol. Acta 14:89. A second type of GTFenzyme(s) is primarily responsible for the synthesis of water-solubleglucose polymers, which consist predominantly of α1-6 linkages. Mutan,containing significant additional α1-3 linkages appears to be importantin the adherence phenomena of S. mutans. Mukasa, H., and H. D. Slade.1973. Mechanisms of adherence of S. mutans to smooth surfaces. I. Rolesof insoluble dextran-levan synthetase enzymes and cell wallpolysaccaride antigens in plaque formation. Infect. Immun. 8:555.

S. mutans cells have been used in experiments designed to study theeffects of immunization on experimental dental caries in rodent andprimate model systems. Taubman, M. A. 1973. Role of immunization indental disease. In Comparative Immunology of the Oral Cavity. Edited byS. Mergenhagen and H. Scherp. U.S. Government Printing Office,Washington, D. C. P. 138. Taubman, M. A., and D. J. Smith. 1974. Effectsof local immunization with Streptococcus mutans on induction of salivaryIgA antibody and experimental dental caries in rats. Infect. Immun.9:1079. McGhee, J. R., S. M. Michalek, J. Webb, J. M. Navia, A. F. R.Rahman, and D. W. Legler. 1975. Effective immunity to dental caries:protection of gnotobiotic rats by local immunization with Streptococcusmutans. J. Immunol. 114:300. Bowen, W. H., B. Cohen, M. F. Cole, and G.Colman. 1975. Immunization against dental caries. British Dent. J.139:45. Lehner, T., S. J. Challacombe, and J. Caldwell. 1975.Immunological and bacteriological basis for vaccination against dentalcaries in Rhesus monkeys. Nature 254:517. Evans, R. T., F. G. Emmings,and R. J. Genco. 1975. Prevention of Streptococcus mutans infection oftooth surfaces by salivary antibody in irus monkeys; (Macacafasicularis). Infect. Immun. 12:293. In these experiments the use ofwhole cell antigens, which often bear GTF on the surface, has resultedin diminished colonization of S. mutans or reduced dental cariesformation. Immunized animals often contained demonstrable serum and/orsalivary antibody to GTF in addition to antibody of other specificities.Emmings, F. G., R. T. Evans; and R. J. Genco. 1975. Antibody response inthe parotid fluid and serum of irus monkeys (Macaca fasicularis) afterlocal immunization with Streptococcus mutans. Infect. Immun. 12:281;Russell, M. W., S. J. Challacombe, and T. Lehner. 1976. Serumglucosyltransferase-inhibiting antibodies and dental caries in Rhesusmonkeys immunized against Streptococcus mutans. Immunology 30:619;Genco, R. J., R. T. Evans, and M. A. Taubman. 1974. Specifically ofantibodies to Streptococcus mutans; significance in inhibition ofadherence. Adv. Exp. Med. Biol. 45:327.

The use of materials containing GTF enzymatic activity as antigens has,at times, also resulted in caries reductions in preliminary experimentsusing the rodent model. Hayashi, J. A., I. L. Shklair, and A. N. Bahn.1972. Immunization with dextransucrases and glycosidic hydrolases. J.Dent. Res. 51:436. However, similar experiments in primates have seldomshown reductions. Lehner, T., S. J. Challacombe, and J. Caldwell. 1975.An immunological investigation into the prevention of caries indeciduous teeth of Rhesus monkeys. Arch. Oral Biol. 20:305.

In vitro experiments have demonstrated that antibody to GTF caninterfere with the formation of the polysaccharide product as well asreduce the adherence of S. mutans to hard surfaces such as wire orglass. Fukui, K. Y. Fukui, and T. Moriyama. 1974. Some immunochemicalproperties of dextransucrase and invertase from Streptococcus mutans.Infect. Immun. 10:985. Therefore, the significance of GTF in themanifestation of S. mutans virulence, the ability to immunologicallyinhibit GTF enzyme function, and the presence of antibody directed toGTF in animals protected against caries, implicated these enzymes aspotential antigens for the study of the effects of immunization onexperimental dental caries.

A number of vaccines have been proposed for immunization against dentalcaries in animals. Various of these proposals are summarized in U.S.Pat. No. 3,879,545. The invention claimed in that patent relates tocaries-preventive vaccines incorporating as the active ingredientthereof a polyfructan (or levan) polysaccharide produced by elaborationof certain strains of streptococcus, particularly Streptococcus StrainSS2. The immunization technique, according to the patentee, has beenfound to result in the formation of antibodies against the heterogeneousmicro-organisms in the recticuloendothelial system and in the blood, andto result in significant decreases in the formation of caries lesions inhost animals subjected to innoculation with such organisms. However, thepatentees also disclose investigating the use of dextransucrase enzymefor immunization against caries formation.

As disclosed in U.S. Pat. No. 3,879,545, the patentees had believed thatsince the enzymes dextransucrase and levansucrase are produced by S.mutans and S. Strain SS2, respectively, and are involved in thesynthesis of dextran and levan, and these polysaccharides are believedto produce caries, that immunization with these enzymes might result inthe production of antibodies to the enzymes which would neutralize theiractivity in vivo, thus inhibiting synthesis of the polysaccharides andresulting in decreased plaque formations and lowered incidence ofcaries. The patentees disclose, however, that their experimentationsfailed to confirm this hypothesis. Nevertheless, the data disclosed didappear to show that while enzyme preparations purified as described byGuggenheim and Newbrun in Helv. Odontol. Acta. 13:84-97(1969) wereineffective in two routes of systemic immunization, a more crude formwas somewhat effective against infectious inoculations with S. mutans6715.

In U.S. Pat. No. 3,931,398 there is disclosed a locally administeredvaccine containing dextransucrase (glucosyl transferase). As disclosedin the patent, this somewhat impure enzyme was prepared by the method ofGuggenheim and Newbrun, supra, from the supernatant liquid of an 18-hourculture of S. mutans 6715 grown on 8% sucrose. These preparations, asdisclosed in the patent, had 10 units of dextransucrase activity per mg.of protein, one unit of the enzyme being defined as the amount requiredto catalyze transformation of 1 mg. of sucrose to dextran in 1 hour(releasing 0.52 mg. of fructose) at pH 6.8. The activity was measured bydetermining the amount of released reducing sugars. While the patenteespeculates that the use of purer forms of the dextransucrase enzymewould result in further diminution of the mean caries scores disclosed,no method is disclosed in this patent of obtaining a purer form of theenzyme.

SUMMARY OF THE INVENTION

In accordance with one aspect of the present invention, the invention isbased upon certain methods for recovering the enzyme glucosyltransferase from Streptococcus mutans in various stages of purification,and to the use of these purer forms of the enzyme in local immunizationagainst dental caries.

The first method of purifying GTF involves the further purifications bygel filtration of the somewhat impure, purified GTF obtained by themethods of Guggenheim, B., and E. Newbrun. 1969. Extracellularglucosyltransferase activity of HS strain of Streptococcus mutans. Helv.Odontol. Acta 13:84, and which is disclosed in U.S. Pat. No. 3,931,398.

A second method of purification according to the invention involves anenzyme antigen preparation obtained by chromatography of dialyzed,concentrated S. mutans culture supernatants on diethylaminoethyl(DEAE)--cellulose, followed by gel filtration.

Defined enzyme antigens (DE-1 and DE-2) were prepared in a third methodof purification from concentrated S. mutans culture supernatant byDEAE--cellulose chromatography followed by gel filtrations, these beingthe two GTF fractions obtained after filtration. These are used inaccordance with the invention as separate antigens.

In the synthesis of mutan, a portion of GTF-remains non-covalently boundto the polysaccharide. A further method of obtaining more purified GTFaccording to the invention involves the recovery of the GTF from thepolysaccharide by use of a denaturing solvent. This method differs quitedistinctly from the other methods of the invention, and from the priorart of which we are aware in that the enzyme is recovered from thepolysaccharide product, rather than from the culture supernatant.

Quite advantageously, in accordance with a further aspect of theinvention, it has been discovered that

(1) Glucosyltransferase serotypes a and g are closely relatedantigenically but are more distantly related to GTF of serotype c, basedon assays of inhibition of total glucan synthesis,

(2) Local immunization with GTF from serotype c or serotype g orserotype a S. mutans reduces the colonization, caries, and lesionscaused by infection with the respective homologous strain compared withsham-injected controls; and

(3) Local immunization with GTF of serotype c or serotype g or serotypea S. mutans reduces the colonization, caries, and lesions caused byinfection with heterologous S. mutans of serotype g (strain 6715), orserotype c (Ingbritt) or serotype g (6715), respectively.

DETAILED DESCRIPTION OF THE INVENTION AND THE PREFERRED EMBODIMENTSEXAMPLE 1 GROWTH OF CULTURE MEDIUM AND CRUDE ANTIGEN PREPARATION

Streptococcus mutans strain 6715, a cariogenic bacteria in rats andhamsters, originally isolated from humans and resistant to streptomycinat concentrations of 2000 μg/ml. was grown anaerobically (10% CO₂, 90%N₂) for 18 to 36 hr at 37° C. in 6 to 10 liters of dialyzate of BrainHeart Infusion medium (BHI) available commercially from BaltimoreBiological Laboratories (BBL). This dialzyable medium had beensupplemented with 0.8% glucose and 3 g/liter of K₂ HPO₄, Carlsson, J.,E. Newbrun, and B. K. Krasse. 1969. Purification and properties ofdextransucrase from Streptococcus sanguis. Arch. Oral Biol. 14:469.Glucosegrown cells were used since these conditions maximize the amountof GTF in the culture supernatant, Spinell, D. M., and R. J. Gibbons.1975. Influence of culture medium on the glucosyltransferase and dextranbinding capacity of Streptococcus mutans 6715 cells. Infect. Immun.10:1448. After removal of the cells by centrifugation (18,000×G) theculture supernatant was concentrated by negative pressure at 4° C.

EXAMPLE 2 PURIFICATION OF GTF BY GEL FILTRATION

The concentrated culture supernatant in Example 1 was subjected tohydroxylapatite chromatography after the technique of Guggenheim andNewbrun, supra, to obtain a somewhat impure dextransucrase (GTF).Enzymatic activity which formed waterinsoluble polysaccharide was thenfiltered on a column of Sepharose 4B. One peak of GTF activity wasobserved to elute with a relative elution volume (REV=V_(e) /V₀, whereV_(e) =elution volume and V₀ =void volume) of 2.1 and formed a cloudy,fine product upon incubation with 0.125 M sucrose for 4 hr. Glucoseaccounted for less than 20% of the total reducing sugar released duringpolysaccharide synthesis as determined by the Somogyi (Somogyi, M. 1945.A new reagent for the determination of sugars. J. Biol. Chem. 160:61)and Glucostat (Worthington Biochemical Corp., Freehold, N.J.) assays.The enzyme-containing fraction (antigen No. 1-CE-1) was shown ashereafter disclosed to contain at least three antigens, one of which wasenzyme when tested in gel diffusion and immunoelectrophoretic analysisagainst a rabbit antiserum to the antigen. No serotype-specificcarbohydrate antigen was detected, either in this preparation withpurified antibody directed to the S. mutans 6715 antigen, (Iacono, V.J., M. A. Taubman, D. J. Smith, and E. C. Moreno. 1976. Aspectrophotometric procedure for quantitation of antibody directed tobacterial antigens. Immunochemistry 13:235), or in any of the followingenzyme preparations described in Examples 3 and 4. In addition, theserums of rats and hamsters immunized with these antigen preparationsdid not react with purified serotypespecific carbohydrate antigen of S.mutans 6715, Iacono, V. J., M. A. Taubman, D. J. Smith, and M. J.Levine. 1975. Isolation and immunochemical characterization of thegroup-specific antigen of Streptococcus mutans 6715. Infect. Immun.11:117.

EXAMPLE 3 PURIFICATIONS OF GTF BY DEAE-Cellulose CHROMATOGRAPHY AND GELFILTRATION

Preparations were obtained by chromatography of dialyzed, concentratedS. mutans culture supernatants according to Example 1 ondiethylaminoethyl (DEAE)-cellulose in accordance with usual techniques.All water-insoluble polysaccharide-forming enzymatic activity was theneluted by stepwise addition of 0.2 M NaCl and 0.5 M NaCl in 0.01 Mphosphate buffer (PB) pH 6.8, or with a gradient to 0.5 M NaCl in PB.This activity was then conventionally filtered on a column of Sepharose4B. All fractions free of serotype antigen which synthesizedpolysaccharide when incubated with 0.125 M sucrose were combined andreferred to an Antigen No. 2 (CE-2). This material included that whicheluted at the void volume (REV=1) and that which eluted later in theprofile (REV=2).

The percentage of glucose released during polysaccharide synthesis whenthe antigen was incubated with sucrose was similar to that of AntigenNo. 1. Gel diffusion analyses of Antigen No. 2 against the serums ofconventional rats immunized with either antigen showed at least two orthree components. At least one identical component and often two weredetected by these serums and the serums of gnotobiotic rats (G)immunized with Antigen No. 2 or hamsters (H) immunized with DE-1 or DE-2as later more fully disclosed. The G serums detected at least four orfive components in gel diffusion analysis and the H serums at leastthree or at least five components, respectively. Enzyme to be used forthe gnotobiotic rat experiment was filter-sterilized.

EXAMPLE 4 TWO GTF FRACTIONS OBTAINED FOR USE AS SEPARATE ANTIGENS

Defined enzyme antigens were prepared from concentrated S. mutansculture supernatant by DEAE-cellulose chromatography followed bySepharose 4B gel filtration techniques also used in the preparation ofAntigen No. 2. However, after gel filtration two GTF fractions (DE-1 andDE-2) were obtained for use as separate antigens. DE-1 eluted at thevoid volume and synthesized a flocculant water-insoluble product uponincubation with sucrose. Fructose was the principal sugar released(76%). This amount of glucose release (24%) can be directly attributedto GTF enzymatic activity of S. mutans strain 6715. Ciardi, J. E., G. J.Hageage, Jr., and C. L. Wittenberger. 1976. Multicomponent nature of theglucosyltransferase system of Streptococcus mutans. J. Dent. Res. 55(Special Issue C):C87. Little polysaccharide was observed after additionof ethanol, to 75%, to the centrifuged supernatant. Polyacrylamide gelelectrophoresis on 7% gels in tris (hydroxymethyl) amino-methane-glucinebuffer (pH 8.8) was performed. Davis, B. J. 1964. Disc electrophoresisII. Methods and application to human serum proteins. Ann. N.Y. Acad.Sci. 121:404. Samples were applied in glycerol directly onto theseparating gel surface. After electrophoresis (4 mA/gel) for 45 min thegel was sliced longitudinally; one-half was incubated in 0.125 M sucroseat 37° C. for 16 hr and the other half was stained with Amido-black(5%). It was seen that the one band which entered the gel correspondedto the single band of insoluble polysaccharide formed by the enzyme fromsucrose. Protein and GTF activity also remained at the origin.

The second GTF fraction, DE-2, eluted from the Sepharose 4B column witha relative elution volume of 1.9. Although some finely dispersedwater-insoluble polysaccharide was synthesized by DE-2, considerablepolysaccharide was observed after addition of ethanol, to 75%, to thecentrifuged supernatant. Fructose was the principal sugar released(83%). Many stained bands could be observed after polyacrylamide gelelectrophoresis. However, no definite band of water-insolublepolysaccharide could be seen in the gels, after 16 hr incubation withsucrose.

DE-1 contained at least one and DE-2 at least three antigenic componentsidentifiable by gel diffusion analyses against rat antiserums to AntigenNo. 2. Gel diffusion analyses of the serums of hamsters immunized withDE-1 vs the DE-1 and DE-2 preparations revealed at least two antigeniccomponents in DE-1, at least one of which was immunologically identicalto one component of at least four detected in DE-2. Hamster antiserumsto DE-2 detected at least three components in Antigen No. 1, at leastone of which was immunologically identical to one of at least fourcomponents detected in DE-2. After immunoelectrophoresis (IEP) of the DEantigens, at least one component demonstrating the same mobility in eachpreparation was shown to have enzyme activity after placement of sucrosein the trough. A second enzyme component was seen in DE-1. The maximumnumber of components seen after IEP with either serum to DE-1 was atleast three and, in DE-2, at least eight.

EXAMPLE 5 IMMUNIZATION AND INFECTION

Two immunization experiments (P) were performed in initiallypathogen-free rats (CD strain, Charles River), in addition to oneexperiment (G) in initially germfree rats (Forsyth strain) and oneexperiment (H) in NIH-white hamsters. Both rat strains were initiallyderived from Sprague-Dawley rats. The rats and hamsters did not harborS. mutans indigenously.

In experiment P1, 38 pathogen-free rats were divided into four groups:(I) nonimmunized and noninfected, (II) nonimmunized and infected, (III)sham-immunized with 0.1 ml phosphate-buffered saline (PBS) incorporatedinto 0.1 ml of complete Freund's adjuvant (CFA, Difco Laboratories,Detroit, Mich.) and infected; (IV) immunized with 0.1 ml enzymeincorporated into 0.1 ml CFA and infected. In experiment P2, 59pathogen-free rats were divided into three groups (groups I, III, IV).

Immunization was initiated when the animals were 21 (P1) or 20 (P2) daysof age. The animals in groups III and IV were injected subcutaneously at7- to 12-day-intervals in the vicinity of the parotid and submandibularglands five times before infection. Rats in group IV of experiment P1were injected with 0.1 mg of Antigen No. 1 which contained 1.8 units ofactivity (unit=the amount of enzyme converting 1 mg sucrose to glucan in1 hr. releasing 0.52 mg fructose). Koepsell, H. J., and H. M. Tsuchiya.1952. Enzymatic synthesis of dextran. J. Bacteriol. 63:293. Rats ingroup IV of P2 were injected with 0.5 mg of Antigen No. 2 whichcontained 2.0 units of activity. Subsequent injections were given at 20-to 30-day intervals after infection. Weanling rats for these experimentswere derived from mothers which had been maintained on a low fluoridelow carbohydrate diet L-356 (General Biochemicals, Chagrin Falls, Ohio)throughout pregnancy until weaning. Experimental rats were continued onthis diet until the week preceding infection when they were given diet2000 which was continued until experimental termination. Keyes, P. H.,and H. V. Jordan. 1964. Periodontal lesions in the Syrian hamster. III.Findings related to an infectious and transmissible component. Arch.Oral Biol. 9:377

The experiment initiated in germfree (G) rats followed essentially thesame protocol as the P experiments. Since dental caries does not occurin germfree conditions only two groups of animals were used,corresponding to groups III and IV. These animals were housed in thesame isolator. At 24 days of age the rats in group IV were given aseries of three injections of Antigen No. 2 (0.5 mg containing 2 unitsof activity/rat) incorporated into CFA administered before infection (53days of age). Rats in group III were injected on the same days with CFAand PBS. Three rats from groups III and IV were removed from theisolator at 53 days for analysis of serum and saliva. Periodic swabbingof the animals in the isolator during the experiment revealed theabsence of microorganisms before infection and only S. mutans 6715 afterinfection. Rats in the G experiment were maintained on cariogenic diet2000 from weaning until the experiment was terminated.

Hamsters were immunized with the defined enzyme preparations (DE-1 andDE-2). The general treatment of hamsters in groups I and III wasidentical to the comparable groups in the rat experiments. Immunizedhamsters were divided into two groups: (IV A) injected with 0.1 ml ofDE-1 (containing 1.6 units of activity) incorporated into 0.1 ml CFA,and (IV B) injected with 0.1 ml of DE-2 (containing 1.6 units ofactivity incorporated into 0.1 ml of CFA. Four injections were given at7-day-intervals in the salivary gland vicinity (SGV) before infectionand one injection was given midway in the 39-day infection period. Allhamsters were maintained on Purine Lab Chow, in meal form, from weaningto 3 days before infection when diet 2000 was initiated.

S. mutans strain 6715 organisms were passaged in conventional ratsmaintained on diet 2000. These organisms were reisolated periodicallyfor purposes of infection. The rats and hamsters in groups II, III andIV were orally infected with 0.4 ml of a 20 hr culture of S. mutans 6715(approximately 10⁸ colony-forming units, (C.F.U.) 10 to 11 days aftercompletion of the initial immunization regimen. Hamsters were infectedon days 52 and 53; P1 rats: Days 74 and 79; P2 rats: Days 68,69, 103,104, 109 and 110; and G1 rats: Days 53. Before this time salivary (andserum) GTF-inhibiting activity could be demonstrated in all theimmunized animals. The flora of all animals was periodically monitoredafter infection as described previously. Taubman, M. A. and D. J. Smith,1974. Infect. Immun. 9:1079.

At the termination of the experiment (39 to 119 days after infection)saliva was collected and the animals were exsanguinated.Streptomycin-resistant S. mutans 6715, total organisms, caries andlesions were determined in P1 as previously described. Taubman andSmith, supra. In experiments P2, G1 and H1, bacteriologic data wereobtained by removing all bacterial plaque from the buccal and lingualsurfaces of the teeth with a number 6 explorer. Plaque was thensonicated for 10 sec at maximum amplitude in 2 ml of one-fourth strengthRinger's solution, appropriately diluted and plated on mitis-salivarius(MS) agar and MS agar containing 200 μg streptomycin/ml (MSS). The wholejaws were then defleshed and all caries and lesions were scored by themodified Keyes method, Taubman and Smith, supra, without knowledge ofthe group designation of the animal.

Differences among means were determined by analysis of variance.Individual means were also compared by analysis of variance. In allexperiments comparisons were made between the immunized group IV and thesham-immunized group III or between the untreated control group I andthe sham-immunized group III. The results are tabulated below.

Salivas and serums were collected and treated before antibody assay aspreviously described. Taubman, M. A. and D. J. Smith. 1974. Infect.Immun. 9:1079. In addition, saliva to be used in the inhibition of ¹⁴C-glucose incorporation assays was dialyzed, first against PBScontaining 0.001 M EDTA, then against 0.01 M phosphate buffer, pH 6.8.

EXAMPLE 6 PREPARATION OF ANTISERUMS

The preparation and monospecificity of rabbit anti-rat secretory IgAserum has been previously described. Taubman, M. A. 1973. U. S. G. P.O., Taubman, M. A. and D. J. Smith, Infect. Immun. 9:1079, both supra.Before use the globulins from this serum were precipitated with ammoniumsulfate at 33% saturation, washed three times with 40% saturatedammonium sulfate and reconstituted to one-fifth the original serumvolume in PBS. Quantitative precipitin analyses with the anti-rat IgAreagent showed that 10 μl was in excess of the amount necessary to reactwith all the IgA in 140 μl of saliva from normal or from hyperimmunizedrats.

The preparation of a rabbit antiserum directed to rat IgG has also beenpreviously described in the immediately preceding noted publications.This antiserum reacted with rat IgG1 and IgG2 and also had light (L)chain reactivity.

Before adsorption, the globulins from this serum were precipitated withammonium sulfate at 33% saturation, washed three times with 40%saturated ammonium sulfate and reconstituted to the original serumvolume in PBS. This anti-rat IgG globulin was treated with animmunoadsorbent prepared by reacting cyanogen bromide-activatedSepharose 6B (Axen, R. J., Porath, and S. E. Ernback. 1967. Chemicalcoupling of peptides and proteins to polysaccharides by means ofcyanogen halides. Nature 214:1302.) with a 20 hour pepsin digest(Utsumi, S., and F. Karush. 1965. Peptic fragmentation of rabbit γG-immunoglobulin Biochemistry 4:1766) of DEAE-cellulose purified rat IgGand/or treated with an immunoadsorbent prepared from cyanogenbromide-activated Sepharose 4B and an IgA-rich fraction of rat colostrum(prepared by gel filtration through tandem columns of Sephadex G-200 andSepharose 6B). Subsequent gel diffusion and immunoelectrophoreticanalyses of the adsorbed globulin showed no reactivity with ratsecretory IgA or IgM and reactivity with rat IgG. Quantitativeprecipitin analyses with the pooled monospecific anti-rat IgG globulinshowed that 50 μl was in excess of the amount necessary to precipitateall the IgG in 5 μl of hyperimmunized or normal rat serum.

Goat anti-rabbit IgG was prepared as previously described or waspurchased (Miles Laboratories, Kankakee, IL).

EXAMPLE 7 PREPARATION OF RADIOACTIVE GTF ENZYME ANTIGENS

Sterile reconstituted protein hydrolysate (³ H) (5 mCi Schwartz/Mann,Orangeburg, N.J.) was added to 100 ml of the diolyzable BHI medium.Fifty milliliters of 10-fold concentrated non-radioactive culturesupernatant were added to radioactive culture supernatant and themixture was dialyzed extensively against PB. This material was subjectedto DEAE-cellulose chromatography, as described for DE preparations,followed by filtration on a column of Sepharose 4B. The material, whichconstituted the GTF antigen used for the radioactive antigen-bindingassay, eluted as a peak of radioactivity with REV of 2 and wascoincident with a peak of enzyme activity. The specific activity of thismaterial was 2180 cpm/μg lyophilized antigen. The material was 32%protein and 68% carbohydrate. Lowry O. H., J. N. Rosebrough, A. L. Farr,and R. J. Randall. 1951. Protein measurement with the Folin phenolreagent. J. Biol. Chem. 193:265, Dubois, M., K. A. Gilles, J. K.Hamilton, P. A. Rebers, and F. Smith. 1956. Colorimetric method fordetermination of sugars and related substances. Anal. Chem. 28.350.After electrophoresis of the antigen (4% polyacrylamide disc gel) andcounting gel slices, more than 70% of the radioactivity of the materialapplied was found in a single peak. Cox, G. S., and T. W. Conway. 1975.Template properties of bacteriophage T4 vegetative DNA. I. Isolation andcharacterization of two template fractions from gently lysed T4-infectedbacteria. J. Biol. Chem. 250:8926. Gel diffusion analyses of thismaterial against the serums of G rats immunized with Antigen No. 2 orhamsters immunized with Antigens DE-1 or DE-2, revealed only a singleidentical antigenic component.

EXAMPLE 8 ASSAY FOR INHIBITION OF GTF ACTIVITY

Two separate assays were used to detect antibody activity in the serumsand salivas of rats immunized with Antigens Nos. 1 and 2. The firstassay was used to demonstrate functional inhibition of GTF-medatedglucose incorporation into total polysaccharide. Both serums and salivasof rats and hamsters taken prior to infection and at the termination ofthe experiment were analyzed. Before infection, the serums of ratsimmunized three or five times or hamsters immunized four times showedmean enzyme inhibition of from 52.4% to 59.2%. At termination, after theanimals had been immunized 1 to 5 additional times, the level of seruminhibition was maintained in one experiment (P1), diminished about 10%in two experiments (P2, G1) and substantially raised in the hamsterexperiments. Both hydroxylapatite-prepared (Antigen No. 1) and theDEAE-cellulose-prepared antigens, (Antigen No. 2) elicited similarlevels of inhibitory activity in the rat experiments. The serums ofsham-immunized rats and hamsters showed no significant effect onenzymatic activity.

Salivas of pathogen-free and gnotobiotic rats immunized with Antigen No.1 also inhibited polysaccharide formation by GTF. Inhibition was from7.9% to 12.3% with immune salivas taken before infection and from 5.3%to 30.6% with salivas taken at experiment termination. Althoughreductions in ¹⁴ C-glucose incorporation into ethanol-insolublepolysaccharide were low, immune (group IV) salivary inhibition wasalways greater than that occurring in the salivas of sham-immunizedanimals (group III), in both rat models. In experiment P1,sham-immunized animals' saliva taken at experimental termination showed12.5% inhibition of GTF activity. This may be due to the extensiveperiod of infection in this experiment (119 days) which may haveresulted in production of salivary antibody to the infecting organism.In the hamster saliva both defined enzyme antigens (DE-1 and DE-2)elicited measurable GTF-inhibiting activity following four injections inthe salivary gland region. The difference between the mean inhibition bysalivas of hamsters injected with DE-1 or DE-2 was not statisticallymeaningful. Inhibition was seen when these salivas were assayed againstammonium sulfate precipitated enzyme, against crude enzyme obtained byion exchange chromatography and gel filtration, or against definedenzyme (DE-1).

The procedure for determining inhibition of glucosyltransferase activityby serum and saliva, a modification of the method of Evans and Genco(Evans, R. T., and R. J. Genco. 1973. Inhibition of glucosyltransferaseactivity by antisera to known serotypes of Streptococcus mutans. InfectImmun. 7:237), has been described previously (Smith, D. J., and M. A.Taubman. 1977. Antigenic relatedness of glucosyltransferases fromStreptococcus mutans. Infect. Immun. 15:91-103). GTF activity wasmeasured by determining ¹⁴ C-glucose incorporation from glucosyl-labeledsucrose into ethanol-insoluble polysaccharide which contains bothwater-insoluble and ethanol-insoluble polysaccharide. Inhibition of GTFactivity was expressed as the percentage reduction in amountsincorporated into precipitated polysaccharide by enzyme in the presenceof immune serums (salivas) compared with incorporation by enzyme in thepresence of control serums or salivas (usually from group I; group IIIin experiment G1). The mean counts ± standard error (S.E.) incorporatedby various enzyme preparations in the presence of control serums rangedfrom 1315±52 to 4494±170 in the experiments described herinafter. Forall experiments, the mean percentage difference of the countsincorporated in the presence of individual control serums from the meanof all control serums was 4.9%. The mean counts ± S.E. incorporated byenzyme in the presence of control saliva ranged from 2987±73 to8594±289. For all experiments the mean percentage difference of thecounts incorporated in the presence of individual control salivas fromthe mean of all control salivas was 4.3%.

Inhibition of the two enzyme preparations used for assay (ammoniumsulfate precipitated culture supernatant enzyme or Antigens as disclosedearlier) by the same serums from immunized rats and hamsters, did notdiffer by more than 2.4%. That inhibition in these assays reflectsspecific antibody activity has been demonstrated by using purified IgGanti-GTF antibody. Russell, M. W., S. J. Challacombe, and T. Lehner;Smith, D. J. and M. A. Taubman, both supra. In addition, specificprecipitation and removal of IgA immunoglobulin from immune saliva leftinsignificant inhibitory activity. Genco, R. J., R. T. Evan, and M. A.Taubman, supra.

EXAMPLE 9 RADIOACTIVE GTF ANTIGEN BINDING ASSAY

The second assay used to detect antibody was the measurement of theradioactive antigen-binding capacity in serum and saliva (Table Ibelow). This assay was used to confirm the antibody nature of theGTF-inhibitory activity in rat serum and saliva and to determine theclasses of antibody of this specificity in saliva. Since previousstudies showed that the principal S. mutans agglutinating antibody inrat serum was of the IgG class (Taubman, M. A., and D. J. Smith Infect.Immun. 9:1079, supra.), rat serum IgG GTF-binding activity wasprecipitated with excess rabbit-anti-rat IgG. Little binding ofradiolabeled GTF was observed by serums of control rats in pathogen-freeor gnotobiotic rat models. However, serums of antigen-immunized ratsdisplayed significant GTF binding which could be attributed to IgGantibody. Salivas were also assayed for binding activity using excessrabbit antiglobulin reagent directed to rat IgA. Antibody of the IgAclass, directed to GTF could be detected in immune rat salivas bothbefore infection and at experiment termination. Although the bindinglevels were low, values were always higher than sham-immunized controls.IgG antibody was also detected in salivas of immunized gnotobiotic ratson day 53 and day 93(191±33 ng and 162±32 ng GTF bound/ml,respectively), indicating that these enzyme antigens elicit salivaryantibody in both IgA and IgG classes when the rats are immunized in thesalivary gland region. In general, the levels of GTF inhibition(functional assay) in both serum and saliva could be related to thelevels of radioactive GTF bound, e.g., in experiment G1 the serums takenbefore infection showed 66.9% and 51.5% inhibition and 225 μg and 242 μgGTF bound/ml of serum, respectively. Salivas taken at the same timeshowed 295 ng and 13 ng GTF antigen bound by IgA/ml, and 223 ng and 158ng GTF antigen bound by IgG/ml saliva. Inhibitions by these salivas were8.4% and 7.3%, respectively.

Levels of binding of the radiolabeled GTF preparation were highest withserums or salivas from experiement G1. The radioactive antigen used forassay was prepared at the same time as the CE-2 preparation used forimmunizing the gnotobiotic rats. Other serums from animals which hadbeen immunized with other similar antigen preparations obtained in alike manner, were analyzed retrospectively, i.e., after storage forperiods up to 1.7 years. These factors may account for the lower levelsof binding in experiments P1 and P2.

                                      TABLE I                                     __________________________________________________________________________    Specific Radiolabeled-GTF Binding by IgG or IgA                               from Serum and Saliva of Rats                                                     Day of         Serum (μg GTF)                                                                      Saliva (ng                                        Experi-                                                                           Collec-                                                                           Group      bound by GTF bound by                                      ment                                                                              tion                                                                              (N) Antigen                                                                              IgG/ml).sup.a                                                                          IgA/ml).sup.a                                     __________________________________________________________________________    P1  71  III(4)                                                                            CFA + PBS                                                                            NT.sup.b 0 ± 0                                                  IV(10)                                                                            CFA + CE-1                                                                           NT       67 ± 18                                            193 III(6)                                                                            CFA + PBS                                                                            2.8 ± 2.4                                                                           NT                                                        IV(11)                                                                            CFA + CE-1                                                                           67.2 ± 14.7                                                                         NT                                                P2  64  III(6)                                                                            CFA + PBS                                                                            NT       15 ± 7                                                 IV(19)                                                                            CFA + CE-2                                                                           NT       28 ± 21                                            168 III(6)                                                                            CFA + PBS                                                                            1.3 ± 0.6                                                                           10 ± 5                                                 IV(22)                                                                            CFA + CE-2                                                                           16.1 ± 1.9                                                                          14 ± 3                                         G1  53  III(1)                                                                            CFA + PBS                                                                            0        0                                                         IV(2)                                                                             CFA + CE-2                                                                           248.8 ± 6.5                                                                         154 ± 141                                          93  III(6)                                                                            CFA + PBS                                                                            1.2 ± 0.5                                                                           8 ±  3.sup.c                                           IV(6)                                                                             CFA + CE-2                                                                           219.3 ± 22.8                                                                        54 ± 15.sup.c                                  __________________________________________________________________________     .sup.a Expressed as group mean ± standard error.                           .sup.b NT, not tested                                                         .sup.c Five salivas tested in each group on this occasion, (N=5)?        

The antigen-binding capacities of serum and saliva samples and therelative contribution of IgG and IgA were determined by a modificationof the method described by Waldman and Henney. (Waldman, R. H., and C.S. Henney. 1971. Cell-mediated immunity and antibody responses in therespiratory tract after local and systemic immunization. J. Exp. Med.134:482) Excess radioactive antigen was added to 5 μl of the test serum.After incubation at 37° C. for 4 hr, an excess (50 μl) of therabbit-anti-rat IgG globulin reagent was added which caused totalprecipitation of the IgG. The precipitates were incubated overnight at4° C. and were washed three times with cold PBS. The precipitates weresolubilized with 0.25 N NaOH, neutralized and counted in Ready-SolvSolution VI. The counts per minute recorded were corrected for normalserum controls (serums from animals in group I) and tabulated as theamount of antigen (micrograms) that was bound by IgG in 1 ml of serum.

Individual saliva samples (140 μl) were incubated with an excess ofradioactive antigen at 37° C. for 3 hr, after which 5 μl of normal serumwas added to provide IgG for coprecipitation with the salivary IgG. Theremainder of the procedure is identical to the procedure for the serumIgG-binding determination described above. Salivary IgA-binding wasdetermined on separate samples to which the radioactive antigen had beenadded (37° C. for 3 hr) followed by an excess (10 μl) of therabbit-anti-rat IgA globulin and incubation at 4° C. overnight. Noprecipitate was visible. Sufficient goat anti-rabbit IgG serum was thenadded to precipitate the rabbit IgG antibody which had bound the ratsalivary IgG-GTF complexes. These reagents were incubated at 37° C. for2 hr and at 4° C. for 2 hr. The ensuing precipitates were treated asdescribed above. Knowledge of the specific activity of the GTFpreparation permitted calculation of the nanograms of antigen bound byIgA antibody per milliliter of rat saliva. The reproducibility of theserum IgG radioactive antigen-binding assay was tested by repeating theassay more than 2 months apart. The mean percentage of differencebetween salivary replicates was 2.6% and 6.1% for IgA and IgG,respectively.

EXAMPLE 10 BACTERIAL STUDIES

Since antibody activity in serum and saliva could be raised by localinjection of rats or hamsters with CE-1 or CE-2 or defined enzymeantigens, it was of interest to determine the effect of thisimmunization on the colonization and pathogenesis of cariogenicstreptococci. Therefore, all groups but group I control animals in eachmodel were infected with a strain of S. mutans 6715 which was bothcariogenic and streptomycin resistant. The extent of S. mutans infectionwas qualitatively estimated by systematic swabbing of molar surfaces ofinfected P, G and H experimental animals at intervals throughout theinfection period. In all experiments, fewer S. mutans were recoveredfrom immunized rats or hamsters on all but one swabbing occasion (TableII). The numbers of colony-forming bacterial units at experimentaltermination, determined either by grinding the half jaw (P1) or bybuccal and lingual plaque removal (P2, G1, H1) are also shown in TableII. In each experiment, the mean numbers in group IV (CE-immunized)showed reduction in the number of S. mutans when compared to thenonimmunized group II or to group III. However, in no experiment werethe S. mutans colony-forming units derived from the dental surfaces ofimmune animals statistically lower when compared to the other infectedgroups. Thus, as seen previously when formalin-killed S. mutans wereused for injection (Taubman, M. A. and D. J. Smith. 1974. Infect. Immun.9:1079, supra) the results are only suggestive of reductions inplaque-associated S. mutans recovered from immunized animals. In the oneexperiment in which saliva was analyzed (G1) there were fewer S. mutansin group IV saliva than in group III. This could reflect agglutinationof bacteria by immune saliva. However, the conditions employed forsonication rupture the non-covalent forces by which antibodyagglutinates cells.

                                      TABLE II                                    __________________________________________________________________________    Bacterial Recoveries from Rats and Hamsters During Infection                  and at Experimental Termination                                               During Infection    Experimental Termination                                  Occasions when               Total                                            S. mutans re-       S. mutans                                                                              Colonies ×                                 coveries by × 10.sup.5                                                                      10.sup.6                                                     swabbing.sup.a from                                                           group IV were                                                                              Days                                                             lower than from                                                                            after                                                         Exp.                                                                             group III/total                                                                            infec-                                                                            Geo      Geo                                              No.                                                                              occasions                                                                              Group                                                                             tion                                                                              mean                                                                             (Range)                                                                             mean (Range)                                     __________________________________________________________________________    P1 2/3      II      827                                                                              (15-5330)                                                                           1850                                                                            (365-9950)                                                 III 119.sup.b                                                                         612                                                                              (185-6680)                                                                          479                                                                             (21-8710)                                                  IV      338                                                                              (15-4210)                                                                           454                                                                             (120-8500)                                     P2 2/2      III 100.sup.c                                                                         8  (0.8-43)                                                                            4 (0.08-14)                                                  IV      6  (0.1-112)                                                                           2 (0.01-39)                                      G1 2/2      III  40.sup.d                                                                         19 (8-31)                                                             IV      7  (1-25)                                                 H1 2/2      III  39.sup.c                                                                         167                                                                              (133-221)                                                                           29                                                                              (22-38)                                                    IVA     102                                                                              (31-382)                                                                            18                                                                              (4- 60)                                                    IVB     123                                                                              (43-315)                                                                            20                                                                              (8-52)                                         __________________________________________________________________________     .sup.a Procedure was performed 18, 30 and 52 days (P1), 7 and 38 days         (P2), 7 and 30 days (G1) and 4 and 23 days (H1) postinfection.                .sup.b Colony-forming units (CFU)/half jaw. Total colonies recovered on       blood agar plates.                                                            .sup.c CFU/smooth surface plaque. Total colonies recovered on                 mitissalivarius agar plates without streptomycin.                             .sup.d Only S. mutans 6715 recovered. Mean S. mutans CFU × 10.sup.5     recovered per milliliter of saliva; group III, 27 (3-168), group IV, 10       (3-60).                                                                  

EXAMPLE 11 CARIES SCORES AND LESIONS

Previous studies in the P and G rat models have shown that the use offormalin-killed S. mutans cells as antigen could confer a measure ofprotection from dental disease caused by subsequent infection withcariogenic S. mutans cells as antigen could confer a measure ofprotection from dental disease caused by subsequent infection withcariogenic S. mutans. Taubman, M. A., and D. J. Smith, immediatelypreceding. To assess the effects of injection of CE and DEglucosyltransferase preparations derived from these organisms on suchinfection, the mean caries scores of each group in the P, G, and Hexperiments were determined (Table III). The immunized group IV alwayshad lower mean caries scores than the other infected group or groups.These differences were statistically significant in three of the fourexperiments, including one experiment with each animal model employed.Hamsters immunized with either of the defined antigen preparationsshowed a somewhat higher level of protection than did CE-injected rats(to the limited extent of comparability of these animal models), and asimilar level of protection when compared with each other. Weights ofanimals in experiments P1, P2, and H1 were monitored throughout eachexperiment and were not significantly different among all groups ofanimals on any occasion.

The limited level of caries found in non-infected groups (group I) ofthe conventional rat experiments was comparable to the level seenpreviously in rats maintained on a low carbohydrate diet (L-356) duringthe preinfection period. In one experiment (P2) the caries scores ofimmunized animals were slightly lower than the scores of uninfectedanimals. This difference was not statistically meaningful nor was itfound when the lesions were examined (Table IV). As in the conventionalrats, the hamsters showed a background of dental caries in noninfectedcontrols. The uninfected group in the H experiment demonstrated aboutone-third of the caries of comparable group I rats. In the hamsters, theduration of infection was only 39 days and the animals' age attermination was 90 days. This infection period, however, was adequate toproduce extensive caries in the sham-immunized (group III) hamsters.Differences in the extent of caries between P1 and P2 in the infectedgroup are most likely the result of differences in the cariogenicity ofthe S. mutans used for infection.

The immune groups of the three different rodent models alwaysdemonstrated lower mean numbers of lesions than the other infectedgroups (Table IV). These reductions were statistically significant whencompared with the other infected groups in three of four experiments.Similarities in the numbers of lesions in uninfected group I andCE-immunized group IV of the P rat experiments are reminiscent of thepattern of protection seen for rats immunized with whole organisms wherethis pattern was suggested to reflect interference with initiation ofnew lesions after infection. Taubman, M. A. and D. J. Smith, supra. Theoccurrence of lesions in the hamsters does not exactly follow thispattern because of the increased virulence of S. mutans in this hamstermodel and the lower background caries. However, it is clear that bothimmunized groups of hamsters demonstrated significantly fewer cariouslesions than comparable sham-immunized controls. Therefore, theinitiation of new lesions has been interfered with to some extent. TheDE-1 antigen might be considered to elicit a slightly more protectiveimmune response than DE-2 since the lesions seen in DE-1-injectedanimals were fewer than those seen in DE-2-injected animals (both ofwhich were significantly lower than adjuvant-injected hamsters).

                                      TABLE III                                   __________________________________________________________________________    Means Caries Scores of Immunized and Control                                  Rats and Hamsters                                                             Treatment                                                                              Group  P1(CE-1).sup.a                                                                      P2(CE-2)                                                                            G1(CE-2)                                                                            H1(DE)                                      __________________________________________________________________________    Noninfected,                mean caries                                       nonimmunized                                                                           I      12.2±3.4                                                                         12.6±1.4                                                                         scores.sup.b                                                                         4.6±0.7.sup.c                           Infected,                                                                              II     35.3±10.0                                                  nonimmunized                                                                  Infected,                                                                              III    33.0±9.5                                                                         16.7±1.6                                                                         17.8±3.1                                                                         47.3±10.3                                sham-immunized                                                                Infected,       21.9±5.5                                                                          10.9±1.2.sup.d                                                                   7.4±1.1.sup.d                                 immunized                                                                              IV                                                                            IVA(DE-1)                15.4±1.9.sup.c                                    IVB(DE-2)                17.4±2.5.sup.c                           __________________________________________________________________________     .sup.a Experiment (antigen used for immunizing group IV)                      .sup.b Group means and standard errors; each rat group represents the         scores of at least six animals, hamster group III, four animals, other        hamster groups; I (5), IVA (12), and IVB (12). Caries scores were obtaine     by a modified Keyes method, supra.                                            .sup.c Statistically significant, p<0.001                                     .sup.d Statistically signifcant, p<0.01                                  

                                      TABLE IV                                    __________________________________________________________________________    Mean Numbers of Carious Lesions of Immunized and Control                      Rats and Hamsters                                                             Treatment                                                                              Group  P1(CE-1).sup.a                                                                      P2(CE-2)                                                                            G1(CE-2)                                                                            H1(DE)                                      __________________________________________________________________________                                mean nos.                                         Noninfected,                                                                           I      20.0±1.8.sup.c                                                                   17.0±1.0.sup.d                                                                   of carious                                                                          8.2±1.9.sup.e                            nonimmunized                lesions.sup.b                                     Infected,                                                                              II     27.9±2.3                                                   nonimmunized                                                                  Infected,                                                                              III    28.0±4.0                                                                         20.8±1.0                                                                         21.5±2.9                                                                         42.0±6.4                                 sham, immunized                                                               Infected,                                                                     immunized                                                                              IV     24.9±2.4                                                                         17.8±1.0.sup.c                                                                   13.7±1.4.sup.c                                          IVA(DE-1)                21.2±4.5.sup.e                                    IVA(DE-2)                24.3±1.9.sup.f                           __________________________________________________________________________     .sup.a Experiment (antigen used for immunizing group IV).                     .sup.b Group means and standard errors, each rat group represents the         numbers of lesions of at least 6 animals; hamster group III, 4 animals,       other hamster groups: I(5), IVA(12), and IVB(12). Carious lesions,            evaluated by a modified Keyes method, were scored as the number of sites      of disease on each molar surface.                                             .sup.c Statistically significant, p<0.05.                                     .sup.d Statistically significant, p<0.01.                                     .sup.e Statistically significant, p<0.001.                                    .sup.f Statistically significant, p<0.005.                               

The caries scores and lesions of GTF-immunized and control animals weredetermined with respect to surface. The scores and lesions of thesham-immunized Group III animals were then compared with those of thecorresponding Group IV animals by calculating the percentage reductionsof immune vs sham (Table V). Reductions in both parameters were alwaysseen in immunized animals on both occlusal, and smooth (buccal andlingual) surfaces. The smooth surfaces examined displayed the greatestreduction in number of lesions in every experiment. In all experiments,except P2, smooth surface reductions also were predominant when cariesscores were compared. This pattern supports the concept that aninterference with the production of new lesions, primarily on smoothsurfaces, has occurred in immunized animals.

                                      TABLE V                                     __________________________________________________________________________    Reduction of Caries Scores or Lesions on Occlusal                             or Smooth Surfaces of Immunized Rats and Hamsters                             Compared with Control Rats and Hamsters                                                     Percentage Reduction                                                          Caries.sup.a                                                                             Lesions.sup.b                                                           Buccal &   Buccal &                                                      Occlusal                                                                           Lingual                                                                             Occlusal                                                                           Lingual                                         Model    Antigen                                                                            Surface                                                                            Surface                                                                             Surface                                                                            Surface                                         __________________________________________________________________________    P1 Pathogen-                                                                           CE-1 24.2 50.2  10.6 24.7                                               free rat                                                                   P2 Pathogen-                                                                     free rat                                                                            CE-2 36.8 22.9  17.7 44.4                                            G1 Gnotobio-                                                                           CE-2 54.8 100   19.6 100                                                tic                                                                        H1 Hamster                                                                             DE-1 56.5 79.5  39.1 62.8                                                     DE-2 50.5 77.2  31.4 55.9                                            __________________________________________________________________________     .sup.a 100 - [(Mean caries score of group IV)/(mean caries score of group     III) × 100].                                                            .sup.b 100 - [(Mean number of lesions of group IV)/(mean number of lesion     of grup III) × 100].                                               

The experiments set forth in the above examples, (Examples 1-11), havebeen disclosed in The Journal of Immunology, Vol. 118, No. 2, pp710-720, Effects of Local Immunization With GlucosyltransferaseFractions from Streptococcus Mutans on Dental Caries in Rats andHamsters, incorporated herein by reference.

Previous studies have shown that local immunization of eitherconventional or gnotobiotic rats with particulate S. mutans antigens(formalinized whole cells) gave rise to primarily a salivary IgAresponse after local immunization. Taubman, M. A. 1973. Taubman, M. A.and D. J. Smith 1974, both supra. In these studies rats demonstrating asalivary IgA response were shown to have less caries than comparablesham-immunized control animals. In the experiments described in theabove examples, soluble preparations of GTF antigens in various stagesof purification were used for local immunization after incorporationinto complete Freund's adjuvant. In those gnotobiotic animals where theresponse was studied, IgG and IgA salivary antibodies were both presentafter three or five injections of CE. While some have suggested that IgAis the only major Ig in rat saliva, (Bistany, T. S., and T. B. Tomasi,Jr. 1970. Serum and secretory immunoglobulins of the rat.Immunochemistry 7:453), qualtitative studies have indicated that IgA ispresent in slightly higher concentration than IgG2 (McGhee, J. R., S. M.Michalek, and V. K. Ghanta. 1975. Rat immunoglobulins in serum andsecretions: purification of rat IgM, IgA and IgG and their quantitationin serum, colostrom, milk and saliva. Immunochemistry. 12:817. Thus, inany study where CFA is utilized for local stimulation it is notsurprising to find both IgA and IgG antibodies in the saliva of ananimal that has approximately equal amounts of these immunoglobulins.Some of the salivary IgG may be serum derived, but it is clear in therabbit, and probably in the rat, that both the IgG and IgA can belocally synthesized in the salivary glands. (Taubman, M. A., G. G.Emmings and R. J. Genco. 1970. Production of antibodies andimmunoglobulins by rabbit salivary glands. J. Dent. Res. 49 (SpecialIssue):70, Hurlimann, J., and H. Darling. 1971. In vitro synthesis ofimmunoglobulin A by salivary glands from animals of different species.Immunology 21:101.)

It has been our contention that salivary antibody is the most likelyprotective principle in relation to experimental dental caries in rodentmodels. In humans, IgA is the major immunoglobulin in the oral cavity,(Brandtzaeg, P. I. Fjellanger, and S. T. Gjeruldsen. 1970. Humansecretory immunoglobulins. I. Salivary secretions from individuals withnormal or low levels of serum immunoglobulins. Scand J. Haematol.(Suppl.) 12:1), and the suggestion has been made that human IgA canfunction to interfere with the interactions necessary for plaqueformation. Taubman, M. A., and D. J. Smith. 1976. Immune components indental plaque. J. Dent. Res. 55 (Special Issue C):C153. There may be asignificant correlation between high caries experience and a low IgAsecretion rate. Orstavik, D. and P. Brandtzaeg. 1975. Secretion ofparotid IgA in relation to gingival inflammation and dental cariesexperience in man. Arch. Oral Biol. 20.701. Furthermore, recentexperiments of Michalek and her colleagues (Michalek, S. M., J. R.McGhee, J. M. Mestecky, R. R. Arnold, and L. Bozzo. 1976. Ingestion ofStreptococcus mutans induces secretory immunoglobulin A and cariesimmunity. Science 192:1238) demonstrated that salivary antibody alonecan be protective with respect to dental caries. Only a salivary IgAresponse could be detected when gnotobiotic rats were fed killed S.mutans whole cells, as we have also observed. Taubman, M. A., and D. J.Smith. 1973. Induction of salivary IgA antibody in rats and hamsters. J.Dent. Res. 52 (Special Issue):276. In the absence of detectable serumantibody significant reductions in dental caries were shown afterinfection with S. mutans. Michalek, S. M., J. R. McGhee et al. supra.Therefore, it appears that salivary antibody, alone, can be protectivewith regard to dental caries.

In the rodent model described in the above examples, it would seemreasonable to assume that the combination of salivary IgG and IgAantibodies functions in a manner similar to IgA in human saliva. Inthese rodent experiments we have not only shown binding of radioactiveglucosyltransferase (GTF) enzymes by serum and salivary antibody, but wehave also shown that immune serums and salivas could inhibit thefunction of these enzymes. Some contention exists as to the ability ofnonimmune serum or oral fluid (Burckhardt, J. J., and B. Guggenheim.1976. Interactions of antisera, sera and oral fluid withglucosyltransferases. Infect. Immun. 13:1009, and Russell, M. W., S. J.Challacombe, and T. Lehner, supra.) to enhance GTF activity, whencompared to enzyme preparations in buffer, in the assay we haveemployed. Although such may be the case, due to interaction andstabilization or possibly primer function, it is clear that if propercontrols are included, this assay is sensitive, reproducible andmeasures antibody. Also of interest is the suggestion that humansecretory IgA which is not antibody to GTF, may enhance the activity ofsome types of GTF. Fukui, K. Y. Fukui, and T. Moriyama. 1974.Acceleration of dextransucrase activity of Streptococcus mutans bysecretory immunoglobulin A. J. Bacteriol. 118:805. Purified ratsecretory IgA does not significantly enhance GTF activity in the assaywe routinely perform. Inhibition of the function of GTF enzymes isparticularly important since these enzymes have been implicated in theexpression of the pathogenic potential of cariogenic S. mutans. Evidencestrongly suggests that the ability of this microorganism to adhere tothe tooth surface, and participate in the formation of dental plaques,depends on the synthesis of extracellular glucose polymers from sucrose.Gibbons R. J. and J. van Houte, supra. Recently, Michalek and herco-workers (Michalek, S. M., T. Shiota, T. Ikeda, J. M. Navia, and J. R.McGhee. 1975. Virulence of Streptococcus mutans: Bio chemical andpathogenic characteristics of mutant isolates. Proc. Soc. Exp. Biol.Med. 150:498) have been able to relate the synthesis of water-insolubleglucan (by GTF) and in vitro adherence directly to the cariogenicity(virulence) of S. mutans mutants. They showed that mutants, synthesizingincreased amounts of water-insoluble polysaccharide, demonstratedincreased adherence and greater cariogenicity than wild type organisms.These studies emphasize the highly significant role of the GTF of S.mutans in the expression of their virulence.

Previously we have suggested that two likely antigens of S. mutans forimmunization experiments might be either the serotype-specificcarbohydrate antigen or the GTF enzymes. Smith, D. J., and M. S.Taubman. 1976. Immunization experiments using the rodent caries model.J. Dent Res. 55 (Special Issue C):C193. This suggestion was based on thedemonstration that antibody directed to either of these antigens had thecapacity to interfere with adherence phenomena demonstrated by S. mutansin vitro. Mukasa, H., and H. D. Slade. 1974. Mechanism of adherence ofStreptococcus mutans to smooth surfaces. II. Nature of the binding siteand the adsorption of dextran-levan synthetase enzyme on the cell wallsurface of the streptococcus. Infect. Immun. 9:419, Iacono, V. J., M. A.Taubman, D. J. Smith, P. R. Garant, and J. R. Pollock. 1976. Structureand function of the type-specific polysaccharide of Streptococcus mutans6715. Immunology Abstracts (Special Suppl.):75. Although both antigensoccur in culture supernatants, in the current invention, we have beencareful to eliminate type-specific antigen from all our GTF preparationsused for immunization. Antibody reactive with the type-specific antigenwas never detected in the serums of any of the animals immunized withthese enzyme preparations. The levels of protection reported in theabove examples, utilizing CE-1 and CE-2 GTF as an antigen in the ratmodels, are quite comparable to the levels of protection obtainedpreviously after immunization with whole cells. Taubman, Smith, supra.The experiment in hamsters with more defined GTF enzymes (DE-1 and DE-2)as immunogens also supports the contention that GTF enzyme is of majorimportance as antigen. Although the evidence is not unequivocal, thereare several additional compelling reasons in support of the case for GTFenzyme: (a) Other enzyme antigens (e.g. fructosyltransferase orinvertase) were probably absent from our GTF fractions. (b) Both GTFenzyme preparations, having only one enzyme antigen in common, gave riseto protection. (c) Although the DE-1 preparation contained trace amountsof material reactive with an antiserum directed to the polyglycerolphosphate (PGP) backbone of teichoic acid, as did DE-2, serum antibodyfrom hamsters immunized with either of these preparations did not reactwith teichoic acid from S. Sanguis. (d) Immunized animals showedantibody in serum and saliva which would bind and inhibit GTF activity.(3) The likelihood of DE-1 and DE-2 containing common antigens otherthan enzyme is low due to the complex series of procedure followed andthe purposeful selection for material demonstrating enzyme (GTF)activity. Nevertheless, it is clear that even more purified enzyme asantigen would better establish the importance of GTF in the pathogenesisof S. mutans and also the importance of GTF as antigen for immunization.

The use of purified antigens for immunization with S. mutans is alsoimportant for other reasons. Van de Rijn and his colleagues (Van deRijn, I., A. S. Bleiweis, and J. B. Zabriskie. 1976. Antigens inStreptococcus mutans cross reactive with human heart muscle. J. Dent.Res. 55(Special Issue C):C59.) have apparently demonstrated, by indirectimmunofluorescent staining, that the serums of rabbits inoculatedintravenously with S. mutans contain antibody reactive with humanmyocardium. The type of fluorescent staining appears to resemble thatseen both after injection of rabbits with group A streptococcalmembranes and with acute rheumatic fever patients' serums. Indeed, humanheart reactive antibody in the serums of S. mutans immunized rabbitscould be removed by adsorption with group A streptococcal membranes.Although the significance of this type of antibody has never beenestablished, and no direct evidence exists that these antibodies arecytotoxic, it is conceivable that antibody directed to S. mutansorganisms could play a role in the pathogenesis of rheumatic fever.Although it is quite unlikely that GTF is an important antigen in theinduction of heart reactive antibody, (group A streptococci do notpossess these cell-bound enzymes), serums from animals immunized withGTF will have to be examined for the presence of heart reactiveantibody.

The exact mechanism whereby antibody can interfere with the molecularpathogenesis of dental caries caused by S. mutans is not completelyunderstood. However, the following suggestions are consistent withcurrent knowledge: We will consider that there are essentially two typesof GTF enzymes (one type synthesizing water-soluble product and theother type synthesizing water-insoluble product). In the presence ofsucrose, enzyme synthesizing water-soluble product initiates synthesisof dextran-like polymer extracellularly. Mukasa, H. and H. D. Slade.1973, 1974, both supra. It is clear that a receptor(s) exist on S.mutans which can specifically bind dextran (Spinell, D. M. and R. J.Gibbons, 1975; Mukasa, H. and H. D. Slade, 1974, and Iacono, V. J., M.A. Taubman, D. J. Smith, P. R. Garant, and J. R. Pollock, supra. Dextransynthesized by GTF can bind to the receptor(s) for dextran. GTF, whichcan also bind dextran, can then join the cell-associated GTF-dextrancomplex. Since sucrose is present, the GTF can then initiate synthesisof mutan (Water-insoluble product) by using the dextran as a primer foradded glucose molecules or by direct synthesis. Germaine, G. R., A. M.Chludzinski, and C. F. Schachtele. 1974. Streptococcus mutansdextransucrase: requirement for primer dextran. J. Bacteriol. 120:287.The active synthesis (Mukasa, H. and H. D. Slade, supra) of mutan isnecessary for the organism to manifest adherence to hard surfaces.Disruption of dextran synthesis could also affect the ability of S.mutans to agglutinate. This parameter may also be of significance indental plaque formation. Therefore, intereference with either dextran ormutan-synthesizing GTF by antibody should theoretically result inreductions in the bacterial masses adherent to the teeth. This in turnwould result in less secretion of acid endproducts and less toothdemineralization. Such reductions in caries compared to appropriatecontrols were found in all experiments in which animals were immunizedwith GTF.

While the suggestion has been made that the water-insolublepolysaccharide (mutan) is more significant in S. mutans pathogenesisthen the soluble polysaccharide (Michalek, S. M., T. Shiota, T. Ikeda,J. M. Navia, and J. R. McGhee, supra.), and our data in the H1experiment suggest a higher level of protection with DE-1 antigen, it isclear that antibody elicited by the DE-2 preparation is also protective.Therefore, at this stage it would seem to be premature to exclude one oranother type of GTF activity from consideration as antigen.

Recently we have concluded from studies of the antigenic relatedness ofGTF enzymes, using numerous immune serums and salivas, that two or atmost three antigenically distinct subsets of GTF enzymes exist among theserotypes of S. mutans. Smith, D. J. and M. A. Taubman, 1977, supra.Therefore, it is conceivable that enzyme preparations from a limitednumber of representative serotypes could be used to provide protectionagainst all serotypes. Furthermore, it may be possible to present theseantigens in such a fashion as to provide stimulaion of cellular elementsboth in minor salivary glands (Crawford, J. M., M. A. Taubman, and D. J.Smith. 1975. Minor salivary glands as a major source of secretoryimmunoglobulin A in the human oral cavity. Science 190:1206) and ingut-associated lymphoid tissue (Michalek, S. M., J. R. McGhee, J.Mestecky, R. R. Arnold and L. Bozzo, supra), in order to elicit aprotective response.

Immunization with somewhat crude glucosyltransferase preparations hasresulted in significant reduction in experimental dental caries inrodents. However, these preparations, derived from culture supernatantshave usually contained both water-soluble and water-insoluble glucansynthetic activity, together with several non-enzyme antigens. In orderto identify GTF as the critical antigen in in vivo protection andcrossprotection, and to evaluate the protective effect of GTF which isresponsible for the synthesis, of water-insoluble glucan, the followingtechnique was devised for GTF preparation.

EXAMPLE 12 PURIFICATION OF GLUCOSYLTRANSFERASE ANTIGEN BY RECOVERY FROMPOLYSACCHARIDE

To prepare GTF synthesizing mutan, i.e., water insoluble glucan,cultures of Streptococcus mutans serotype a (strain E49), serotype c(strain Ingbritt) or serotype g (strain 6715) were grown in dialyzed BHImedium containing 1% glucose or a completely defined synthetic mediaalso containing glucose for 20 hrs. at 37° C. The cells were removed bycentrifugation according to usual techniques, and the culturesupernatants were then incubated at 37° C. with 10% sucrose at pH 6.5,together with 0.02% sodium azide and 0.04 M salts. The water-insolublepolysaccharide in each supernatant formed was collected bycentrifugation, washed (6 X PBS; 6×dist. H₂ O (and incubated at 4° C.for 1 hr. with stirring in one to two times its volume in 6 Mguanidine-HCl, to remove the portion of GTF which synthesizes mutan andwhich remains non-covalently bound to the polysaccharide. Thepolysaccharide was then removed by centrifugation, the guanidine wasremoved from the supernatant by dialysis with 0.05 M Na phosphate andthe GTF in the supernatant was then enriched by gel filtration, ashereinafter disclosed.

Other denaturing solvents other than guanidine-HCl can be used, ifdesired, for example, urea and sodium dodecylsulfate. However, guanidineHCl has been found quite satisfactory in the practice of the invention.A relatively high concentration of solvent should be used, but from apractical aspect, a concentration of from 1:1 to 2:1 is most desired.

EXAMPLE 13 TIME COURSE OF THE RELEASE OF ENZYMATIC ACTIVITY

To determine the time course of the release of enzmatic activity whenwater-insoluble polysaccharides are exposed to guanidine-HCl, separate10 gm polysaccharide samples were incubated with the denaturant for timeperiods ranging from 10 nutes to 70 hours. After removal of thepolysaccharide, enzymatic activity in the dialyzed supernatants wasdetermined by the release of total reducing sugars and glucose afterincubation with sucrose. Little change occurred in the amount ofactivity released from strain E49 polysaccharide at the various timestested. Activity released from polysaccharide of strain 6715 increaseduntil 20 hours and plateaued thereafter. Strain Ingbritt showed thegreatest rate of change in release of GTF activity. Increasing amountsof activity were released through approximately 30 hours of incubation.

In each strain, the ratio of dextransucrase units released/ml., releasedat 1 and 20 hours, was similar to a ratio of the amount of glucansynthesized (determined radioisotopically) to these two times,indicating that the chemical assays were indeed reflecting GTF activity.Thus, GTF is released from water-insoluble polysaccharide from the threeserotypes tested, albeit at differing rates. This difference may bedependant on chain length, amount or extent of branching, or the varietyand ratio of linkages in the respective polysaccharides, as well asvariations in the enzymes themselves.

EXAMPLE 4 RATIO OF CARBOHYDRATE TO PROTEIN RELEASED DURING EXPOSUREPOLYSACCHARIDE TO GUANIDINE

Since guanidine might also have a disruptive effect on the structure ofpolysaccharide itself, samples were also analyzed for Lowrey protein andfor carbohydrate by the method of Dubois, both supra. Accordingly, theratio of CHO to protein released at various time intervals for strain6715 was determined. At relatively short periods of incubation, it wasfound that protein represents one half to two thirds of the material inthe supernatant. However, carbohydrate becomes the predominant componentreleased at incubation periods longer than about 8 hours.Immunodiffusion analyses of this supernatant with purified antibodydirected to glucan of the homologous strain, suggested that at least aproportion of this CHO is glucan.

EXAMPLE 15 ENRICHMENT OF GTF FROM S. MUTANS 6715

S. mutans strain 6715 GTF was selected for enrichment since this straindemonstrated little fructosyltransferase activity. After dialysis andconcentration, the GTF-containing supernatant was gel filtered accordingto usual techniques on 8% agarose. Enzymatic activity in the elutionswas determined by the Somogyi assay, supra. All enzymatic activityeluted (optical density 280 nm) at the void volume of the 8%agarose-column. The enzyme-containing fractions were then gel filteredon 2% agarose. Again most of the material eluted in one peak near thevoid volume and contained the enzymatic activity.

EXAMPLE 16 PROPERTIES OF GTF ENZYME RECOVERED

The properties of the enzyme contained in this peak are tabulated below.

    ______________________________________                                        Properties of 6M GuHCl-Eluted GTF (strain 6715) Following                     Gel Filtration on 8% and 2% Agarose                                           ______________________________________                                        Specific Activity     2.7                                                     (U/mg)                                                                        Total U               5.4                                                     Product               Insoluble                                               GTF/FTF               >97                                                     (Incorporation of .sup.14 C-glucose                                           and .sup.3 H-fructose from sucrose)                                           ______________________________________                                    

The pool had a rather high specific activity. A total of 5.4 Units werecontained in the pool. When enzyme was incubated with sucrose, onlyinsoluble polysaccharide could be detected spectrophotometrically.Radioactive incorporation assays indicated that the polysaccharideformed was virtually all glucan. Thus the enzyme recovered could becharacterized as glucosyltransferase forming only water-insolubleglucan.

The pool was also electrophoresed on duplicate 5% disc gels. Afterelectrophoresis at 4 ma/gel for 1 hour, one gel was stained for proteinwith Amido Black and the other gel was incubated with sucrose todetermine zones of mutan-synthetic activity. Only one band of proteinand one corresponding area of enzymatic activity were seen, bothmigrating the same small distance from the origin.

The 2% agarose pool was also examined for antigenic components inimmunodiffusion. The GTF pool was placed in the central well. Antiserato the serotype g antigen, to teichoic acid, to the glucan of S. mutansstrain 6715, and to a crude enzyme antigen preparation from culturesupernatants of the 6715 strain were placed in the outer wells. Noserotype antigen or teichoic acid could be detected in the enzymepreparation according to the invention. However, a precipitin band didform with the anti-glucan antiserum. The anti-CEA antiserum reactedpredominantly with one component, forming a precipitin band close to theantigen well. This precipitating system migrated inimmunoelectrophoretic analyses to the same region as the water-insolublesynthetic activity, identified in a separate run when sucrose was addedto the trough. This indicated that the band seen in gel diffusionagainst the anti-CEA serum contained GTF. Thus, the guanidine-eluted andgel-filtered GTF pool seems to contain one protein component which isenzyme and one carbohydrate component which is glucan.

In order to determine the effect on enzymatic activity of antibodydirected to this glucan, the serum from a rat injected twice withguanidine-eluted GTF 6715 was tested against the homologous enzymepreparation in a radioisotopic assay of GTF activity. The serum waseither unabsorbed or was absorbed with twice its volume of SephadexG-25, a predominantly α1-6 link-glucan. Both absorbed and unabsorbedantisera showed essentially the same level of inhibition, indicatingthat antibody directed to this type of glucan did not affect GTFactivity as measured in the assay. This was supported by the observationthat rabbit antibody directed against surface glucan of formalin-killedcells 6715 showed no significant inhibition. These results are shownbelow.

    ______________________________________                                        Effect on Anti-Dextran Antibody on Inhibition of                              6M GuHCl-Eluted GTF (GuGTF) Activity                                                                      % Inhibition.sup.b                                Antiserum      Adsorbant    of GuGTF                                          ______________________________________                                        Rat anti-GTF.sub.6715                                                                        none         31.2 ± 4.8                                     Rat anti-GTF.sub.6715                                                                        G25          37.2 ± 4.5                                     Rabbit anti-glucan.sub.6715 .sup.a                                                           none         2.2 ±                                          ______________________________________                                         .sup.a Ig released (pH 2.3) from G 25 following incubation with antisera      directed to formalin-killed S. mutans 6715 cells                              .sup.b Based on .sup.14 C-glucose incorporation from labeled sucrose into     EtOH-insoluble polysaccharide compared to normal sera.                   

The ability of guanidine-eluted GTF to elicit a serum and secretoryimmune response was determined as follows. Fluids from hamsters and ratsinjected with complete Freund adjuvant (CFA) or with CFA plusguanidine-eluted GTF from serotype a E49 or from serotype g 6715 wereevaluated for inhibition of enzymatic activity. Fluids fromadjuvant-injected animals incorporated essentially the same level of ¹⁴C into EtOH-insoluble polysaccharide as did control sera or salivas.However, GTF from either strain gives rise to a significant inhibitoryresponse in serum when tested against the homologous enzyme. Inaddition, sera from these GTF-immunized animals inhibited enzyme fromserologically related strains at least as well as the homologous strain.Importantly, a significant salivary inhibitory response was alsoelicited by this enzyme preparation.

The ease of preparation, the restricted activity and the immunogenicityof purified enzyme population of the invention provides GTF that ishighly satisfactory as a caries protective antigen.

    ______________________________________                                        GTF Inhibitory Activity in Serum and Saliva of Rodents                        Immunized with GTF Eluted from Mutan with 6M GuHCl                                                 Inhibition Assay.sup.c                                   GTF.sup.a                                                                           Antigen                  GTF.sup.b                                                                           Percentage                               Source                                                                              Preparation                                                                              FLUID (N)     Strain                                                                              Inhibition                               ______________________________________                                              CFA        Hamster Sera (9)                                                                            E49   0.1 ± 4.4                                   GTF + CFA  Hamster Sera (10)                                                                           E49   62.1 ± 3.8                                  GTF + CFA  Hamster Sera (4)                                                                            6715  79.4 ± 1.8                            E49                                                                                 CFA        Hamster Saliva (10)                                                                         E49   0.2 ± 1.8                                   GTF + CFA  Hamster Saliva                                                                              E49   12.8 ± 3.4                                  CFA        Rat Sera (6)  6715  1.8 ± 3.6                             6715  GTF + CFA  Rat Sera (2)  6715  34.2 ± 2.6                                  GTF + CFA  Rat Sera (2)  E49   35.0 ± 1.7                            ______________________________________                                         .sup.a GTF (eluted from mutan with 6M GuHCl) and/or complete Freund's         adjuvant (CFA).                                                               .sup.b 55% (NH.sub.4).sub.2 SO.sub.4 precipitated culture supernatant.        .sup.c Inhibition of 14-C glucose incorporation into EtOH-insoluble gluca     compared to incorporation in presence of normal serum or saliva. Mean .+-     standard error.                                                          

Instead of the gel filtration on 2% agarose as above disclosed, a phenylsepharose column, equilibrated in 6 M guanidine HCl was also used. Thisprocedure removes the glucan from the GTF. The material eluted has beenfound highly satisfactory.

Immunization experiments in the rodent model in which antigenicpreparations containing GTF from serotypes b or g have clearlydemonstrated protection from caries caused by infection with homologousstrains of S. mutans. Hayashi, J. A., Shklair, I. L. and Bahn, A. N., J.Dent. Res. 51:436, 1972 and Taubman, M. A. and Smith, D. J., J. Immunol.118:710, 1977. These lines of evidence seem to indicate that antibodydirected to GTF antigens might protect animals from caries caused byorganisms from which the enzyme was derived (homologous). However, theprotection afforded by such immunization against other strains orserotypes of S. mutans (heterologous) is unclear. S. mutans organismsare heterogeneous and are divided into from three to seven groups, basedon serological, genetic or biochemical differences. Bratthall, D. andKohler, B., J. Dent Res. 55:C15, 1976. Differing antigenic andbiochemical features have also been noted among GTF preparations derivedfrom the various serotypes. Genco R. J., Evans, R. T. and Taubman, M.A., Adv. Exp. Med. Biol. 45:327, 1975; Fukui, K., Fukui, Y. andMoriyama, T., Infect. Immun. 10:985, 1974; Linzer, R. and Slade, H. O.,Infect. Immun. 13:494, 1976; Smith, D. J. and Taubman, M. A., Infect.Immun. 15:91, 1977; Kuramitsu, H. and Ingersoll, L., Infect. Immun.14:636, 1976. However, by in vitro techniques antigenic similaritiesseem to exist among GTF from serotypes a, d, g and among GTF fromserotypes b, c, e. The purpose of the present investigation was todetermine to what extent these in vitro relationships might be reflectedin in vivo protection. The protective and cross-protective effects oflocal immunization with GTF from S. mutans strain Ingbritt (serotype c),6715 (serotype g) or E49 (serotype a), on infection with homologousorganisms (serotype c) or infection with heterologous organisms (S.mutans strain 6715, serotype g or strain Ingbritt serotype c or strainE49 serotype a) was explored in the hamster model.

The following examples show the cross-protective aspece ofglucosyltransferase antigens.

EXAMPLE 17 PREPARATION OF GTF

Strain of Streptococcus mutans strains E49 (serotype a), Ingbritt(serotype c) and 6715 (serotype g), known to be cariogenic in hamstersand resistant to streptomycin at concentrations of 200 μg/ml, were eachgrown anaerobically (10% CO₂, 90% N₂ O) for 24 hours at 37° C. in 6 to10 liters of synthetic media. After centrifugation (9000 rpm) eachcell-free supernatant was pH-adjusted to 6.5. Polysaccharide was thensynthesized in each supernatant by the addition of sucrose to 10% andincubation at 37° C. for 48 hours. Bacterial growth was inhibited byaddition of 0.02% sodium azide.

The water insoluble polysaccharide which was formed in each supernatantwas collected by centrifugation (9000 rpm) at 4° C. and washedextensively with cold distilled H₂ O and 0.01 M sodium phosphate, pH 6.8to which 0.02% sodium azide had been added. Glucosyltransferase enzymeswere then eluted from the washed, water-insoluble polysaccharides by onehour incubation (4° C.) with a volume of 6 M guanidine-HCl which wastwice the weight of the polysaccharide, as reported previously. Smith,D. J., Taubman, M. A. and Ebersole, J. L., J. Dent. Res. 15:A132, 1977.Following elution, guanidine was removed by dialysis and, afterconcentration, the eluate was gel filtered on columns of 8% agatose in0.01 M sodium phosphate, pH 6.8.

Enzyme activity from each strain was detected by the Somogyi (Somogyi,M., J. Biol. Chem. 160:61, 1945) and Glucostat (Worthington Biochemical)assays to elute at the void volume. Fructose was the principle sugarreleased with either serotype a (79%) or serotype c (75%) GTF. NeitherGTF preparation contained teichoic acid or the serotype specificantigen, although glucan was detected when assayed with specificantisera in immuno-diffusion. Guanidine-eluted GTF preparations fromeither serotype a or c formed water-insoluble and ethanol-insolublepolysaccharide when incubated with 0.12 M sucrose for 4 hours at 37° C.

EXAMPLE 18 IMMUNIZATIONS AND INFECTIONS

In all experiments, NIH white hamsters were divided into five groups,(1) nonimmunized and noninfected; (II) sham immunized with 0.1 ml PBSincorporated into 0.1 ml CFA and infected with serotype c strainIngbritt (homologous infection); (III) sham immunized and infected withorganisms from serotype g strain 6715 (heterologous infection); (IV)immunized with 0.1 ml Ingbritt GTF (containing 0.8 units of activity-12)in 0.1 ml CFA and homologously infected; (V) immunized with Ingbritt GTFin CFA and heterologously infected. This is hamster experiment 2(H2). Inhamster experiment 3(H3) the injected GTF was from serotype a (E49) andhomologous infection was with strain E49, while heterologous infectionwas with strain 6715 (serotype g). In hamster experiment 4(H4) theinfected GTF was from serotype g (6715) and homologous infection waswith strain 6715 (serotype g) and the heterologous infection was withstrain Ingbritt (serotype c).

Four injections were given at 7 to 10 day intervals in the salivarygland vicinity (SGV) prior to infection and one injection was givenmidway in the 40 day infection period. All hamsters were maintained onPurina Mouse chow from weaning to three days prior to infection whendiet 2000 was initiated. Keyes, P. H. and Jordan, H. V., Archs. OralBiol. 9.377, 1964. The weights of the animals were not significantlydifferent among the five groups of animals for the duration of theexperiment.

The hamsters (H2) in groups II and IV, and groups III and V were orallyinfected with 0.4 ml of 20 hour cultures of S. mutans (approximately 10⁸colony forming units) strains Ingbritt (homologous infection) and 6715(heterologous infection) respectively, 8 days after completion of theinitial immunization regimen. Hamsters in experiments 3 and 4 wereinfected with similar quantities of the heterologous or homologousorganisms described above. Prior to this time salivary (and serum)GTF-inhibiting activity could be demonstrated in all the immunizedanimals. The flora of all animals was periodically monitored by swabbingand plating onto Mitis-Salivarius (MS) agar and MS agar containing 200μg streptomycin/ml (MSS). Taubman, M. A., and Smith. D. J., Infect.Immunity 9.1079, 1974.

At the termination of the experiment saliva was collected and theanimals were exsanguinated. Caries and lesions were scored by a modifiedKeyes method without knowledge of the group designation of the animal,as previously described.

Salivas and sera were collected and treated as previously described.Taubman, M. A. and D. J. Smith. Infect. Immunity 9:1079, 1974. Inaddition, saliva to be used in the inhibition of ¹⁴ C-glucoseincorporation assays was dialyzed, first against PBS containing 0.02 MEDTA, then against PBS.

EXAMPLE 19 IN VITRO ANTIGENIC RELATEDNESS OF S. MUTANS GTF ASSAY FORINHIBITION OF GTF ACTIVITY

Glucosyltransferase activity was measured by determining the amount of¹⁴ C-glucose incorporation into an ethanol insoluble polysaccharide byglucosyltransferase, first preincubated (1 hour) with sera or saliva,then incubated (2 hours) with ¹⁴ C-glucosyl-labelled sucrose and dextranT10 at 37° C. Smith, D. J. and M. A. Taubman, Infect. Immun. 15:91,1977. Inhibition was expressed as the percentage reduction in countsincorporated into precipitated polysaccharide by enzyme in the presenceof immune sera (salivas) compared with incorporation by enzyme in thepresence of control sera or salivas. The results are tabulated below.

Previous studies, as indicated above, have indicated that antigenicdifferences exist among GTF synthesized by various serotypes of S.mutans. These relationships are further explored herein for serotypes a,c and g by determining the extent to which antisera, prepared byinjecting hamsters with GTF eluted from water-insoluble polysaccharideof strains E49, Ingbritt or 6715, would inhibit the formation ofpolysaccharide by GTF in ammonium-sulfate precipitates of culturesupernatants of these three serotypes. Enzyme from serotypes a (E49) andg (6715) are both strongly inhibited by antisera directed to GTF ofeither serotype, indicating that these two enzyme preparations areantigenically related. Enzyme from serotype c (Ingbritt), whileappreciably inhibited by the homologous hamster anti-Ingbritt GTFantisera, is only minimally affected by antisera directed to GTF ofserotypes a or g. Conversely, neither the anti-serotype a or g GTFinhibit the Ingbritt enzyme by more than 12%. Based on these inhibitionpatterns, two subsets of GTF antigens appear to exist within these threeserotypes; one subset includes GTF from serotypes a and g while a secondsubset includes GTF from serotype c. Although enzymes from these twosubsets are antigenically distinct on this basis, the inhibition ofenzyme by antisera of the heterologous subset, while low, is significantcompared with the activity of the enzyme in the presence of sera fromsham-immunized hamsters.

    ______________________________________                                        Inhibition of GTF-mediated .sup.14 C-Glucose Incorporation into ETOH-         Insoluble Polysaccharide by Hamster Anti-GTF Sera                                       Percentage Inhibition of Mean CPM                                             Compared to Normal Sera                                             Hamster     E49        ING        6715                                        Sera.sup.a  GTF        GTF        GTF                                         ______________________________________                                        Anti-E49 GTF                                                                              52 ± 3.sup.b                                                                          8 ± 1   78 ± 3                                   Anti-ING GTF                                                                              12 ± 3  59 ± 2  9 ± 4                                    Anti-6715 GTF                                                                             42 ± 2  12 ± 1  41 ± 3                                   Sham-Injected                                                                             0 ± 2   2 ±0 2  0 ± 2                                    ______________________________________                                         .sup.a Four to 13 sera tested against each enzyme.                            .sup.b Mean ± standard error.                                         

EXAMPLE 20 In Vivo Cross Protection Via Immunization With Serotype c

In order to determine whether the protective effects of GTF immunizationcould be extended across subset boundaries the following experiment wasdevised. Hamsters were either sham-immunized with buffer in CFA orimmunized with serotype c GTF in CFA earlier set forth. AfterGTF-inhibitory activity was demonstrated in sera (mean inhibition ± SE:41.2±1.9) and salivas (11.6±3.6) of enzyme-injected groups, hamsterswere infected with cariogenic and streptomycin resistant strains of S.mutans on day 59. Sham injected group II and Ingbritt GTF-injected groupIV were infected with the heterologous serotype g strain 6715. Duringthe course of the 40 day infection period, animals were swabbed 4 daysand 21 days after infection to determine the effect of immunization oncolonization. The results of these swabbings are presented below. Nostreptomycin-resistant streptococci were detected in the uninfectedgroup I. The geometric mean of the colony forming units of Ingbrittorganisms recovered from GTF-injected group IV were much lower thanIngbritt CFU recovered from the homologously infected, sham-injectedgroup II on days 4 and 21 of infection. These differences werestatistically significant on the second occasion. The geometric mean ofthe colony forming units of 6715 organisms recovered from GTF-injectedgroup V were significantly lower than 6715 CFU recovered from theheterologously infected, sham injected group III on both occasions.Thus, reductions in recoveries or plaque-associated S. mutans of bothhomologous and heterologous serotypes occurred in hamsters immunizedwith the serotype c GTF.

    ______________________________________                                        S.Mutans  Colony Forming Units Recovered from Molar Surfaces of               Hamsters After Infection With Either S.Mutans  Serotype c (Groups             II and IV) or Serotype g (Groups III and V)                                                        Geometric Mean CFU                                                             Infecting                                                                              Day 4.sup.a                                                                          Day 21                                  Group  Treatment/N    Strain   (× 10.sup.-2)                                                                  (× 10.sup.-3)                     I      Noninfected/6  None     0      0                                       II     Sham-immunized/11                                                                            Ingbritt 33.8   238.sup.b                               IV     GTF.sub.ING -immunized/12                                                                    Ingbritt 9.1    17.sup.b                                III    Sham-immunized/12                                                                            6715 j   50.2.sup.c                                                                           773.sup.d                               V      GTF.sub.ING -immunized/12                                                                    6715     4.6.sup.c                                                                            43.sup.d                                ______________________________________                                         .sup.a Days following infection.                                              .sup.b,c Differences between these two groups statistically significant,      p<0.05.                                                                       .sup.d Differences between these two groups statistically significat;         p<0.005.                                                                 

The effect of local injection with serotype c GTF on the disease causedby Ingbritt and 6715 strains was determined by evaluating the caries andlesions on the molar teeth after the 40 day infection period. Theindividual immunized hamster caries scores or lesion counts werecompared with the mean caries scores or lesion counts of the similarlyinfected sham-immunized group. These differences are expressed aspercentage reductions of sham disease. Both caries scores and lesioncounts of GTF-injected hamsters infected with homologous Ingbrittorganisms were reduced by at least 60%. The differences in both theseparameters between the sham group II and immunized group IV weresignificant at the 0.001 level. The caries scores and lesions counts ofGTF-immunized hamsters infected with strain 6715 (Group V) were at least50% lower than the mean caries scores and lesions counts in the shamgroup III infected with the same strain. These differences weresignificant at the 0.025 and 0.001 level respectively.

A comparison of the percentage reductions in disease of the homologouslyinfected group IV and heterologously infected group V revealed thatthere was slightly, but not significantly, greater protection observedin homologously infected groups. In either group, reductions in cariesscores and lesions counts on smooth surfaces were more prominent thanocclusal surface reductions. Thus, the immune response elicited by localinjection of serotype c derived GTF interferes with the pathogenesis,especially on smooth surfaces, of these S. mutans organisms, regardlessof the apparent in vitro antigenic unrelatedness of the GTF which theysynthesize.

In order to determine whether the protective effects of GTF immunizationcould be extended across subset boundaries the following experiment wasdevised. Hamsters were either sham-immunized with buffer in CFA orimmunized with serotype c GTF in CFA earlier set forth. AfterGTF-inhibitory activity was demonstrated in sera (mean inhibition ± SE:53.1±2.5) and salivas 5.2±1.8) of enzyme-injected groups, hamsters wereinfected with cariogenic and streptomycin resistant strains of S. mutanson day 59. Sham injected group II and E49 GTF-injected group IV wereinfected with the heterologous serotype g strain 6715. During the courseof the 40 day infection period, animals were swabbed 4 days and 18 daysafter infection to determine the effect of immunization on colonization.The results of these swabbings are presented below. Nostreptomycin-resistant streptococci were detected in the uninfectedgroup I. The geometric mean of the colony forming units of Ingbrittorganisms recovered from GTF-injected group IV were significantly lowerthan E49 CFU recovered from the homologously infected, sham-injectedgroup II on days 4 and 18 of infection. These differences werestatistically significant on both occasions. The geometric mean of thecolony forming units of 6715 organisms recovered from GTF-injected groupV were significantly lower than 6715 CFU recovered from theheterologously injected, sham injected group III on both occasions.Thus, reductions in recoveries of plaque-associated S. mutans of bothhomologous and heterologous serotypes occurred in hamsters immunizedwith the serotype a GTF.

    __________________________________________________________________________    S.mutans Colony Forming Units Recovered from                                  Molar Surfaces of Hamsters after infection with either                        S.mutans serotype a (Groups II and IV) or serotype g                          (Groups III and V) (H3)                                                       Swabbing             Geometric Mean CFU                                                                       Sacrifice                                                   Infecting                                                       Group                                                                             Treatment Strain                                                                             N Day.sup.a 4                                                                       Day 18 Day 46                                        __________________________________________________________________________    I   Non-infected                                                                            None  6                                                                              0   Not swabbed                                                                          0                                             II  Sham-immunized                                                                          E49  11                                                                              3090.sup.b                                                                        446,683.sup.c                                                                        445                                           IV  GTF.sub.E49 -immunized                                                                  E49  11                                                                              339.sup.b                                                                         26,303.sup.c                                                                         103                                           III Sham-immunized                                                                          6715 11                                                                              56.sup.d                                                                          10,964  91                                           V   GTF.sub.E49 -immunized                                                                  6715 11                                                                              8.sup.d                                                                           589     11                                           __________________________________________________________________________     .sup.a Days following infection.                                              .sup.b,c,d Differences between these two groups statistically significant     b.sub.p <0.001                                                                c.sub.p <0.05                                                                 d.sub.p <0.005                                                           

The effect of local injection with serotype c GTF on the disease causedby Ingbritt and 6715 strains was determined by evaluating the caries andlesions on the molar teeth after the 40 day infection period. Theindividual immunized hamster caries scores or lesion counts werecompared with the mean caries scores or lesion counts of the similarlyinfected sham-immunized group. These differences are expressed aspercentage reductions of sham disease. Caries scores of GTF-injectedhamsters infected with homologous Ingbritt organisms were reduced by atleast 48% and lesion counts by 28%. The differences in these parametersbetween the sham group II and immunized group IV were significant at the0.05 level for caries and 0.005 level for lesion. The caries scorescounts of GTF-immunized hamsters infected with strain 6715 (Group V)were 42% lower than the mean caries scores in the sham group IIIinfected with the same strain. Lesion counts were 25% lower. Thedifferences in lesions were significant at the 0.05 level.

In Vivo Cross Protection Via Immunization With Serotype c

In order to determine whether the protective effects of GTF immunizationcould be extended across other subset boundaries the followingexperiment was devised. Hamsters were either sham-immunized with bufferin CFA or immunized with serotype c GTF in CFA earlier set forth. AfterGTF-inhibitory activity was demonstrated in sera (mean inhibition ± SE:49.0±4.8) and salivas (18±3.5) of enzyme-injected groups, hamsters wereinfected with cariogenic and streptomycin resistant strains of S. mutanson day 59. Sham injected group II and serotype g GTF-injected group IVwere infected with the heterologous serotype c strain Ingbritt. Duringthe course of the 54 day infection period, animals were swabbed 14 daysand 33 days after infection to determine the effect of immunization oncolonization. The results of the swabbings are presented below. Nostreptomycin-resistant streptococci were detected in the uninfectedgroup I. The geometric mean of the colony forming units of 6715organisms recovered from GTF-injected group IV were significantly lowerthan Ingbritt CFU recovered from the homologously infected,sham-injected group II on days 14 and 33 of infection. These differenceswere statistically significant on both occasions. The geometric mean ofthe colony forming units of Ingbritt organisms recovered fromGTF-injected group V were significantly lower than Ingbritt CFUrecovered from the heterologously infected, sham injected group III onboth occasions. Thus, reductions in recoveries of plaque-associated S.mutans of both homologous and heterologous serotypes occurred inhamsters immunized with the serotype g GTF.

    __________________________________________________________________________    S.mutans CFU Recovered by Swabbing (H4)                                                            Geometric Mean CFU                                                                      At Sacrifice                                                   Infecting                                                                          Day 14                                                                             Day 33                                                                             Day 54                                         Group                                                                             Treatment/N Strain                                                                             (× 10.sup.2)                                                                 (× 10.sup.3)                                                                 (× 10.sup.4)                             __________________________________________________________________________    I   Noninjected/7                                                                             None 0    0    0                                              II  Sham-immunized/12                                                                         6715 504* 816**                                                                              736                                            IV  GTF.sub.6715 -immunized/12                                                                6715 87*  123**                                                                              605                                            III Sham-immunized/12                                                                         Ingbritt                                                                           556**                                                                              416***                                                                             516                                            V   GTF.sub.6715 -immunized/12                                                                Ingbritt                                                                           33** 66***                                                                              372                                            __________________________________________________________________________     Differences between sham-and GTF-immunized groups statistically               significant:                                                                  *P<0.01                                                                       **P<0.001                                                                     ***P<0.005                                                               

The effect of local injection with serotype c GTF on the disease causedby Ingbritt and 6715 strains was determined by evaluating the caries andlesions on the molar teeth after the 40 day infection period. Theindividual immunized hamster caries scores or lesion counts werecompared with the mean caries scores or lesion counts of the similarlyinfected sham-immunized group. These differences are expressed aspercentage reductions of sham disease. Both caries scores and lesioncounts of GTF-injected hamsters infected with homologous Ingbrittorganisms were reduced by at least 55%. The differences in caries andlesion these parameters between the sham group II and immunized group IVwere significant at the 0.005 and 0.001 levels respectively. The cariesscores and lesions counts of GTF-immunized hamsters infected with strain6715 (Group V) were at least 56% lower than the mean caries scores andlesions counts in the sham group III infected with the same strain.These differences were significant at the 0.05 and 0.001 levelrespectively.

The usefulness of a caries vaccine is partly dependent upon the extentto which it demonstrates protection against the serotypes of S. mutansnormally associated with human infection. Epidemiologic studies of S.mutans prevalence in human populations have shown that the serotype cstrains predominate among those isolated. Serotype d(g) strains havealso been isolated fairly frequently. These two groups are not onlyserologically distinct but also differ with regard to theirguanine+cytosine contents, cell wall carbohydrates, antigenicrelatedness of their GTF, as shown above, and other biochemical andgenetic features. However, despite these differences, local immunizationwith glucoyltransferase from the serotype c strain elicited a protectiveimmune response against infection with serotype g organisms. Apparentlythe low but significant level of inhibition noted in the in vitro assayreflected an activity which was protective. This could possibly beexplained by the in vivo interference with a critical enzyme function,e.g., insoluble glucan formation, which may not be striking in vitro ifour assay favors soluble glucan formation. Antisera directed againstenzyme synthesizing insoluble glucan has been reported to be a moreeffective inhibitor of GTF from different setotypes than antiseradirected against GTF synthesizing soluble glucan. Linzer, R. and H. O.Slade, and Kuramitsu, H. and L. Ingersoll, both supra. Since the enzymeantigen in the present study was prepared directly from the insolublepolysaccharide, higher levels of more cross-reactive anti-GTF antibodymight be expected to result in enhanced cross-protection.

Previously, primates injected with cell-associated Ingbritt antigens(live, heat-killed or broken cells) showed caries reductions caused byinfection with either Ingbritt or a pre-existent serotype c S. mutansflora. Bowen, W. H., Cohen, B., Cole, M. F. and Coleman, G. Brit. Dent.J. 139:45, 1975, Lehner, T., Challacombe, S. J. and Caldwell, J., Archs.Oral Biol. 20:305, 1975. However, those injected with IngbrittGTF-containing preparations gave little evidence of protection. Whilethis could be explained by a difference in the animal model used forimmunization, a more likely explanation may lie in the physical natureand concentration of the GTF used. In our experiments enzyme is elutedfrom the glucan product with 6M guanidine-HCl. The elution proceduretends to aggregate the enzyme, rendering it potentially moreinnumogenic. In addition, the enzyme is derived from water-insolublepolysaccharide which is generally thought to be more significant thanthe water-soluble form in the pathogenic potential of S. mutans. GibbonsR. J. and van Houte, J., Ann. Rev. Micro. 29:19, 1975. Furthermore, incontrast to the primate studies, GTF-inhibitory activity was present insera and salivas of all injected hamsters prior to infection and theselevels were maintained by booster injection during the infection period.

The results suggest that the formulation of a caries vaccine may requireenzyme antigen from only one strain. However several questions remain,including: (1) whether such cross-protection would occur with GTF fromother strains, (2) which specific GTF component elicits thecaries-protective antibody response, and (3) by what mechanism doesprotection occur.

It will be understood that various changes may be made in the preferredembodiments described hereinabove without departing from the scope ofthe present invention. Accordingly, the preceding should be construed asillustrative and not in a limiting sense.

What is claimed is:
 1. A method of immunization against dental caries,which method comprises:(a) preparing a purified glucosyltransferase(GTF) by culturing Streptococcus mutans in a medium which containsglucose and dialyzable nutrients to provide a culture supernatant,incubating the supernatant with sucrose to synthesize a water-insolublepolysaccharide, recovering the polysaccharide, incubating thewater-insoluble polysaccharide with a denaturing solvent to break thebond between the water-insoluble saccharide and the glucosyltransferase,separating the water-insoluble polysachharide from the denaturingsolvent containing the glucosyltransferase enzyme, separating thedenaturing solvent from the glucosyltransferase enzyme, separating thedenaturing solvent from the glucosyltransferase enzyme in thesupernatant, and concentrating by gel filtration the glucosyltransferaseto obtain an antigen vaccine consisting essentially of the purifiedglucosyltransferase; (b) preparing a vaccine consisting essentially ofthe purified glucosyltransferase; and (c) administering the vaccineobtained through the method of steps (a) and (b) in a sufficient amountlocally into or near the oral cavity of a patient susceptible to dentalcaries, thereby providing for an immunization of the patient againstdental caries.
 2. The method of claim 1 wherein the Streptococcus mutansemployed in preparing the vaccine comprises Streptococcus mutans strain6715, strain Ingbritt or strain E49.
 3. The method of claim 1 whereinthe denaturing solvent employed is a guanidine compound.
 4. The methodof claim 1 wherein the vaccine consisting essentially of GTF is furthercharacterized in having a specific activity of about 2.7 and a GTF/FTFratio of greater than about
 97. 5. The method of claim 1 which comprisesadministering the vaccine locally in the vicinity of the orifices of theduct of the minor salivary glands.
 6. The method of claim 1 wherein theGTF is prepared from a serotype c S. mutans.
 7. The method of claim 1wherein the GTF enzyme is prepared with a serotype c S. mutans andwherein the antigen vaccine is employed in connection with immunizationagainst dental caries caused by S. mutans of the serotype g strain. 8.The method of claim 1 which method comprises administering a vaccinecomprising GTF obtained from Streptococcus mutans strain Ingbritt(serotype c) in sufficient amount to provide a protective immuneresponse against infection with serotype g organisms or serotype corganisms, or GTF from S. mutans strain E49 (serotype a) in sufficientamount to provide a protective immune response against infection withserotype a or serotype g organisms; or GTF from S. mutans strain 6715(serotype g) in sufficient amount to provide a protective immuneresponse against infection with serotype g or serotype c organisms.